Gut Endogenous Protein Flows and Postprandial Metabolic Utilization of Dietary Amino Acids...

301 Pages·2008·3.69 MB·English

by  Amelie Deglaire

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G
ut endogenous protein flows and postprandial
metabolic utilization of dietary amino acids in
simple-stomached animals and humans

A
thesis presented in partial fulfilment of the requirements for the degree of
Doctor of Philosophy in Human Nutrition
at Massey University, Palmerston North, New Zealand

A D
mélie eglaire
2008
♦ i
ABSTRACT

Dietary protein quality depends on two key measures: true ileal protein digestibility and the
metabolic utilization of absorbed amino acids (AA). The objectives of this study were to
determine the influence of two dietary factors (antinutritional factors and peptides) on ileal
endogenous protein flows; to validate the intubation technique used in humans for ileal
digesta sampling; to determine the postprandial metabolic utilization of dietary AA
depending on their delivery form and to assess the validity of the growing pig for predicting
true ileal protein digestibility in the adult human. Investigations were undertaken in the
growing rat, growing pig and adult human. Ileal digesta were collected from euthanised rats,
post valve T-caecum cannulated pigs, and naso-ileal intubated conscious adult humans. Ileal
endogenous nitrogen (N) and AA were measured using a protein-free (PF) diet, diets
containing 15N-labelled casein in the intact (C) or hydrolysed (HC) form, or a diet based on
free AA (diet A), for which some dispensable AA were omitted to allow a direct
determination of their endogenous flows. Digesta centrifugation and ultrafiltration (diet HC)
allowed for the determination of ileal endogenous protein flows and the extent of tracer (15N)
recycling.
Antinutritional factors from a crude extract of kidney beans (Phaseolus vulgaris), when given
at amounts commonly ingested in practice, enhanced ileal endogenous protein flows (rats, PF
diet). After adaptation to the diet, body N balance per se did not influence ileal endogenous
protein flows (rats, diets PF and A) but dietary peptides led to greater ileal endogenous AA
and N flows compared with a protein-free diet. Dietary peptides (HC), compared with
peptides naturally released in the gut during protein digestion (C), did not enhance ileal
endogenous protein flows (rats, pigs, and humans). The extent of tracer recycling, however,
was maximal in frequently-fed rats, lower in meal-fed pigs and minimal in meal-fed humans
(65, 21, and 11% of 15N-labelled ileal endogenous proteins, respectively). Naso-ileal
intubation for ileal digesta sampling in humans was shown to be an accurate method and
evidence was obtained supporting the growing pig as a valid model for predicting true ileal
protein digestibility in the adult human. Finally, the form of delivery of dietary AA (from HC
or C) influenced the postprandial metabolic fate of dietary AA, especially in terms of AA
catabolism kinetics. However, the overall nutritional value of C and HC were similar.
♦ ii
ACKNOWLEDGMENTS

The work reported in this thesis was undertaken in collaboration between the Riddet
Institute (Massey University, Palmerston North, New Zealand) and the UMR914
Nutrition Physiology and Ingestion Behaviour (INRA/AgroParisTech, Paris, France)
under the co-supervision of Distinguished Pr Paul Moughan and Pr Daniel Tomé.

I would like to express my sincere gratitude to Pr Moughan, my chief supervisor, for his
continued guidance and encouragement. The time and energy he has spent on this work is
much appreciated. I would also like to sincerely thank Pr Tomé for initiating this
collaborative work and allowing me to work in his laboratory. I sincerely thank Dr Cécile
Bos (INRA/AgroParisTech, France) for her support and guidance throughout the entire
period of my doctoral study and for her assistance with the human study.

I would like to thank Dr Gordon Reynolds (IFNHH, New Zealand) for performing the
surgical preparations of the pigs. My grateful thanks to Mr Shane Rutherfurd (Riddet
Institute, New Zealand) for all his assistance during the animal experiments and for many
helpful discussions. Thanks to Dr Claire Gaudichon (INRA/AgroParisTech, France) for
having me in her research team. Thanks to Dr Robert Benamouzig (CRNH-IdF, France)
for allowing the completion of the human study in his hospital service. A special thanks
to Dr George Arinei (CRNH-IdF, France) for all the time and energy he spent on the
human study. Thanks also to Mrs Catherine Luengo (INRA/AgroParisTech, France) for
her help during mass spectrometry analysis and to Dr Francois Mariotti
(INRA/AgroParisTech, France) for his helpful suggestions for the validation of the
intubation method. Thanks also to Dr Klaus Petzke (DIfe, Germany), to the staff of the
nutrition laboratory (IFNHH, NZ), in particular Mrs Fliss Jackson and Ms Leiza
Turnbull, for sample analysis. Thanks to the staff of the feed processing unit (IFNHH,
massey University, New Zealand), especially Mr Don Thomas and Mr Kaliwyn Pereka,
for helping with feed preparation. Thanks to Dr Nicole Roy (AgResearch, New Zealand)
for her assistance with setting the pig experiments. Thanks also to Mrs Anne Singh
(Fonterra Ltd., New Zealand) for helping with determination of the casein hydrolysate
molecular weight profile.

Thanks to Dr Alasdair Noble and Dr Patrick Morel for their statistical advice.
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We gratefully acknowledge Mr Jacques Fauquant (INRA, France) for providing 15N-
labelled casein. Thanks to Sanitarium (New Zealand) for providing some cooked wheat
for the pig study. Thanks also to Ajinomoto Co. (France) for providing crystalline amino
acids for the human study.

I would like to thank AgroParisTech, the Riddet Institute and the “fondation de France”
for a grant towards my research.

I would like to acknowledge Zhengrong Yu (INRA/AgroParisTech, France) who assisted
in pig care during her internship and Claire Boutry (INRA/AgroParisTech, France) who
helped during the human study.

Last but not least, I would like to thank all of my friends who have given me so much
support and made these four years enjoyable. A special thanks to my dear Jennie Jean, my
little pidgin friend. Thanks to my office mates, Cheryl for always listening to me, Daniel
for keeping me company during the late evenings at work. I sincerely thank my “proof-
readers”: Aurélie, Liat, Estelle and Karine. Thanks to Romaric, Nicolas, David, Thibaud,
Laureline and Mick for keeping alive the “French connection” in New Zealand. Thanks
to my flatmates, Cam, Saija and Dan for enduring my early or late noise. Thanks to Laure
for always being here while I was in Paris. Thanks also to Clémence and Barbara, for
their encouragement. I would also like to thank my dear Cha for always being present, in
person or virtually. I also thank my parents, all my family members, and Xavier for
always supporting me through the good and difficult stages of this PhD and overseas
experience.
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TABLE OF CONTENTS
Abstract……………………………………………………………………………… i
Acknowledgements………………………………………………………………….. ii
List of Tables……………………………………………………………………………vi
List of Figures………………………………………………………………………..… x
List of abbreviations...………………………………………………...……………..… xii

PREFACE………………………………………………………………………………...1

CHAPTER I REVIEW OF THE LITERATURE………………………………………....3

Introduction………………………………………………………………………………4

I. Evaluation of dietary protein quality in adult humans.…………………………..5
1. Dietary protein and amino acid requirements 5
2. In vivo protein quality index 9
3. Digestibility 13
II. Nitrogen flows along the digestive tract…………………………………………. 19
1. Endogenous nitrogen flows 19
2. Protein digestion and absorption 27
3. Ileal nitrogen losses 30
III. Determination of ileal nitrogen flows…………………………………..………. 33
1. Ileal digesta collection 33
2. Measurement of endogenous nitrogen and amino acid losses 41
IV. Dietary modulation of ileal endogenous nitrogen losses…………………......... 50
1. Dry matter intake 50
2. Dietary protein 51
3. Antinutritional factors 53
4. Dietary fibre 56
5. Dietary electrolyte balance 57
V. Postprandial metabolic fate of dietary nitrogen……………………….…….. 58
1. Regional metabolism and availability of dietary amino acids 59
2. Dietary modulation of postprandial metabolism of dietary nitrogen 65

Conclusions and justification of the experimental work……………………………..72

Literature cited………………………………………………………………………… 75
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CHAPTER II COMMERCIAL PHASEOLUS VULGARIS EXTRACT (STARCH
STOPPER) INCREASES ILEAL ENDOGENOUS AMINO ACID AND CRUDE PROTEIN LOSSES
IN THE GROWING RAT……………………………………………………………….. 115

CHAPTER III FEEDING DIETARY PEPTIDES TO GROWING RATS ENHANCES GUT
ENDOGENOUS PROTEIN FLOWS COMPARED WITH FEEDING PROTEIN-FREE OR FREE
AMINO ACID-BASED DIETS…………………………………………………………... 135

CHAPTER IV A CASEIN HYDROLYSATE DOES NOT ENHANCE GUT
ENDOGENOUS PROTEIN FLOWS COMPARED WITH INTACT CASEIN WHEN FED TO
GROWING RATS……………………………………………………………………... 155

CHAPTER V A CASEIN HYDROLYSATE DOES NOT ENHANCE GUT
ENDOGENOUS PROTEIN FLOWS COMPARED WITH INTACT CASEIN WHEN FED TO
GROWING PIGS……………………………………………………………………... 177

CHAPTER VI VALIDATION OF THE INTUBATION METHOD FOR INTESTINAL
DIGESTA SAMPLING IN CONSCIOUS ADULT HUMANS…........................................…… 202

CHAPTER VII INTACT AND HYDROLYSED CASEIN LEAD TO SIMILAR ILEAL
ENDOGENOUS PROTEIN FLOWS AND SIMILAR POSTPRANDIAL RETENTION OF DIETARY
NITROGEN DESPITE DIFFERENT METABOLIC EFFECTS IN ADULT HUMANS……………. 221

CHAPTER VIII ILEAL DIGESTIBILITY OF DIETARY PROTEIN IN THE GROWING
PIG AND ADULT HUMAN…………………………………………………………….. 253

CHAPTER IX GENERAL DISCUSSION AND CONCLUSION…………………….. 275
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LIST OF TABLES
CHAPTER I
Table 1. Amino acid classification. From Reeds et al. (2000b)……………………... 8
Table 2. Estimates of dietary amino acid requirements……………………………... 9
Table 3. Amino acid scoring pattern across species…………………….................... 10
Table 4. Apparent and true faecal digestibility of nitrogen as estimated in adult
humans and growing rats within controlled studies..…………………………………... 15
Table 5. Ileal endogenous nitrogen flow and its amino acid composition (mean ±
SD) as determined in the rat, pig and human fed a protein-free diet…………………... 17
Table 6. Daily contributions of endogenous nitrogen secretions to total endogenous
nitrogen entering the lumen of the digestive tract in growing pigs. Quoted in Sève et
al. (1997); data from Leterme (1995)………………………………………………….. 19
Table 7. Intestinal proteolytic enzymes. From Le Floch & Sève (2000)……………. 28
Table 8. Amino acid transport systems of the intestine. Adapted from Kilberg et al.
(1993)…………………………………………………………………………………... 29
Table 9. Comparison of the amino acid (AA) composition of ileal endogenous
nitrogen (N) with endogenous nitrogenous secretions in the small intestine of pigs. 32
Table 10.a. Basal ileal endogenous nitrogen and lysine flows as determined in
growing pigs using different methods. ………………………………………………… 42
Table 10.b. Comparison of overall mean (± SD) of published basal ileal endogenous
nitrogen and lysine flows determined in pigs using different methods………………... 43
Table 11. Mean ileal endogenous nitrogen flow as determined in adult humans using
different methods………………………………………………………………………. 43
Table 12. Estimates of portal amino acid balance in young pigs fed liquid-milk
replacer. ………………………………………………………………………………... 61
Table 13. Effect of the molecular form of dietary amino acids [intact protein (IP),
hydrolysed protein (HP), corresponding free mixture (AA)] on the total nitrogen (N)
balance. ………………………………………………………………………………... 69

CHAPTER II
Table 1. Ingredient compositions of the preliminary and experimental diets……… 120
Table 2. Determined amino acid and total nitrogen contents of the essentially
protein-free diet (PF1) and of the crude Phaseolus vulgaris extract (PVE) and
autoclaved Phaseolus vulgaris extract (aPVE)………………………………………… 121
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Table 3. Amylase inhibitor activity, trypsin inhibitor activity and lectin (PHA)
content in the commercial Phaseolus vulgaris extract (PVE) before and after
autoclaving.……………………………………………………………………………. 124
Table 4. Mean ileal endogenous amino acid flows (n = 8) at the terminal ileum of
growing rats fed diets PF1, PF2, PF3, or PF4………………………………………….. 126
Table 5. Mean endogenous amino acid and total nitrogen flows (mg/100 mg dry
matter intake, n = 8) in the faeces of growing rats fed diets PF1, PF2, PF3, and PF4… 127

CHAPTER III

Table 1. Ingredient compositions of the diets given to growing rats…………...…… 139
Table 2. Nutrient and selected mineral contents of the experimental diets given to
the growing rats. ……………………………………………………………………….. 140
Table 3. Endogenous amino acid flows along the digestive tract of growing rats fed
diets PF, A1, A2 and HC. …………………………………………………………….. 144
Table 4. Apparent and standardized ileal amino acid digestibility for growing rats
fed the synthetic amino acid-based diets A1 and A2…………………………………... 145
Table 5. Endogenous ileal amino acid flows determined in rats fed diets PF, A1, A2
and HC. ………………………………………………………………………………... 146
Table 6. Endogenous flows and digestibility of nitrogen at the terminal ileum of
rats fed diets PF, A1, A2 and HC………………………………………………………. 146

CHAPTER IV
Table 1. Ingredient compositions of the diets given to growing rats. ………………. 159
Table 2. Amino acid composition of casein and 15N-enrichment of individual
amino acids.. …………………………………………………………………………... 160
Table 3. Endogenous nitrogen (isotope dilution) expressed as a percentage of total
nitrogen along the digestive tract of rats fed diets C and
HC…………………………………....………………………………………………… 164
Table 4. Ileal total amino acid and nitrogen flows determined in rats fed diets C and
HC. …………………………………………………………………………………….. 165
Table 5. Ileal endogenous amino acid and nitrogen flows determined using the
isotope dilution method in rats fed diets C and HC. …………………………………... 166
Table 6. Ileal endogenous amino acid flows determined using the isotope dilution
or ultrafiltration methods in rats fed diet HC…………………………………………... 167
Table 7. Apparent and standardized ileal digestibility of nitrogen in diets C and HC
fed to growing rats. ……………………………………………………………………. 168
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CHAPTER V

Table 1. Ingredient compositions of the diets fed to growing pigs………………….. 182
Table 2. Amino acid and nitrogen contents of the test diets omitted from diet A…... 183
Table 3. Total ileal amino acid and nitrogen flows in growing pigs fed diets C or
HC after 5-day adaptation to the diets…………………………………………………. 188
Table 4. Ileal endogenous flows of aspartic acid and serine in pigs fed diets C, HC
and A after 5-day adaptation to the test diets …………………………………………. 189
Table 5. Apparent ileal amino acid digestibility for growing pigs fed diet A after 5-
day adaptation to the diet ……………………………………………………………… 189
Table 6. Ileal endogenous amino acid and nitrogen flows in pigs fed diets C, HC or
A after 5-day adaptation to the test diets……………………………………………… 190
Table 7. Ileal endogenous amino acid and nitrogen flows determined using the
isotope dilution or ultrafiltration method in pigs fed diet HC after 5-day dietary
adaptation ……………………………………………………………………………... 191
Table 8. Ileal endogenous amino acid and nitrogen flows in growing pigs fed diets
C, HC or A without prior adaptation to the test diets (acute feeding) ………………… 192

CHAPTER VI

Table 1. Characteristics of the subjects included in the different intubation studies... 206
Table 2. Composition of the meals tested in the different intubation studies………. 207
Table 3. Polyethylene glycol (PEG)-4000 recovery and integrated PEG flows in
ileal digesta for adult humans 8 h postprandially……………………………………… 212

CHAPTER VII
Table 1. Subject characteristics……………………………………………………... 224
Table 2. Ileal endogenous amino acid and nitrogen flows in adult humans fed a
single mixed meal containing intact casein (C, n = 6), hydrolysed casein (HC, n = 5)
or free L-amino acids (A, n =5 ). ……………………………………………………… 233
Table 3. Estimate of dietary 15N recycled within gut endogenous protein (R, %,
molecular weight >10 kDa) determined in ileal digesta after ingestion of a single
mixed meal containing hydrolysed casein (HC, n = 5) in adult humans………………. 234
Table 4. Postprandial metabolic utilization of dietary nitrogen (N) 8 h after
ingestion of a single mixed meal containing intact casein (C, n = 6) or hydrolysed
casein (CH, n = 5) in adult humans……………………………………………………. 239